A highly cost-effective analytical device for performing immunoassays with ultra high sensitivity

ABSTRACT

A simple easy to manufacture analytical device capable of performing membrane based immunoassays on batch of samples within 3 to 10 minutes wherein the method permits focused application of samples, costly labeled immunoassay and signal amplification reagents, said device includes an antibody-immobilized micro porous membrane, breadth corner layer of which is directly attached to a semi-rigid liquid-impervious body with water insoluble adhesive; absorbent body is provided separately and is not attached to analytical device during manufacture, absorbent body is wetted and is placed proximal to the lower surface of the membrane thereby forming networks of capillary channels with the absorbent body; flow of samples or reagents is always kept downwards and focused without application of any force to the absorbent body and the use of disposable adsorbent body permits stepwise addition of signal amplification reagents for ultra sensitive detection of diagnostically important molecules by visual examination of the membrane surface.

FIELD OF THE INVENTION

The present invention relates to analytical device and methods for usein determining analyte in batch of sample suspected of containing suchanalyte with ultra high sensitivity. More particularly, the inventionrelates to device and simple methods for application of signalamplification reagents after immunoassay in a laboratory or a fieldsetting for attaining high sensitivity.

BACKGROUND AND PRIOR ART INVENTION

The diagnostically important molecules may be small molecules ormacromolecules. The small molecules like mycotoxins such as aflatoxinB₁, Ochratoxin A T-2 toxin are present in food and feed such as cerealgrains, ground nuts, rice, peanuts, fodder as contaminants due to fungalinfection. Human and animals are exposed to mycotoxins through ingestionof toxin-contaminated food and feed, inhalation or skin contact. Thedetermination of mycotoxins in agricultural commodities is importantbecause their consumption by man and animals causes mycotoxicosis. Theyalso produce different biological effects such as acute toxicity,mutagenicity, carcinogenicity, tertogenicity etc (Morgan, M. R. A.Tetrahedron 45, 2237, 1989). The determination of the concentration ofsteroids such as estriol, dehydroepiandrosterone sulphate, cortisol,testosterone, hormones such as thyroxin, triiodothyronine and drugs suchas methoxtrate present in biological fluid such as serum, plasma, urinein minute amounts have been accepted as one of the attractive indicatorsof several diseases and in pathological conditions (Methods of HormoneRadioimmunoassay Ed. Jaffe, B. M. and Behrman, H. R. 1979, AcademicPress, New York).

The diagnostically important macromolecules like hormones such asthyroid stimulating hormones (TSH), adrenocortcotropic hormone (ACTH) orcancer markers such as acid phosphatase, ferritin, carcinoembroynicantigen are present in biological fluid such as serum, plasma inextremely minute amounts. These biologically important molecules havebeen widely accepted as the effective indicators of disease. Similarly,diagnosis of various infectious diseases also require detection ofantigen such as hepatitis, malaria or detection of antibodies in casesof diseases like AIDS, amoebiosis (Methods of Enzymatic Analysis, Ed. ByBergmeyer, J. and Gross, vol. X, XI, 1983, Verlag chemie, Weinheim).These antigen or antibodies are present in biological fluid such asserum in extremely minute amounts and early detection sometimes is notpossible. Some of these infectious diseases are detected by the presentavailable methods only at a later stage when the concentration ofantigen or antibody in biological fluid is increased.

Detection of these diagnostically important molecules requires highlysensitive assay system. Immunological assays have proven to be of greatvalue for detection and quantification of numerous analytes in liquidsamples. Because the results of immunological and other specific bindingreactions are frequently not directly observable, various techniqueshave been devised for their indirect observation. Such techniquesinvolve labeling of one of the members of the specific binding pair witha radioisotope, chromophore, fluorophore or enzyme label. Radiolabels,chromophores and fluorophores may be detected by the use of radiationdetectors, spectrophotometers. Where members of a specific binding pairare tagged with an enzyme label, their presence may be detected by theenzymatic activation of a reaction system wherein a compound such as adyestuff, is activated to produce a detectable signal. Such proceduresare described in a number of articles and texts, an example of which isReviews on Immunoassay Technology, Ed. S. B. Pal, Pub. Chapman and Hall,1988.

Among the different immunoassays, enzyme labels tagged with specificbinding pair are widely used. These assays are called enzymeimmunoassay, commonly termed as ELISA. At present for small moleculescompetitive ELISA and for macromolecules sandwich ELISA method is widelyused for their determination (Porstman, T. and Kiessig, S. T. JournalImmunological Methods 150, 5, 1992)

In competitive ELISA, sample or standard is added to a solid phase suchas 96-well microtitre plate, tubes, beads coated with an antibody raisedagainst small molecules to be determined. They are incubated after theaddition of an enzyme, which is covalently linked with the smallmolecule at a temperature in the range of 4° C. to 37° C. for a periodof 2 to 24 hrs. The analyte to be detected competes with a labeledreagent of the same analyte for a limited number of antibody bindingsites. The amount of labeled antigen, which binds to the antibody, isinversely proportional to the amount of the unknown antigen in thesample.

The solid phases are washed with a buffer and substrate solution isadded which gives colour due to the enzymatic activity of the enzymeconjugate bound to the antibody immobilized over solid phase. Theintensity of the colour is inversely proportional to the concentrationof small molecule present in the sample. Visualization of the intensityof the colour with naked eye in comparison with known concentration ofsmall molecule gives an idea about the relative amount of small moleculepresent in the sample. The amount of colour developed may also bemeasured for quantitative determination by spectrophotometer orautomatic microtitre plate reader. The measured absorbances are plottedagainst known concentration of small molecule to obtain a standardcurve. Concentration of small molecule in samples is calculated fromthis standard curve by standard procedure.

In another assay, known as sandwich ELISA method, a solid surface suchas 96-well microtitre plate, tubes are coated with monoclonal antibodyobtained against macromolecules to be determined. Vacant sites areblocked with blocking protein such as casein, BSA. Standards or samplescontaining macromolecules are added to solid phase and incubated at atemperature in the range of 4 to 37° C. for a period of 2 to 24 hrs. Theselected macromolecular antigen in the sample or standard binds to thereceptor monoclonal antibody. The solid phase is washed and furtherincubated for a period of 2 to 24 hrs with a second polyclonal antibody(against macromolecules) covalently linked with an enzyme which iscapable of binding to the bound antigen to form an immobilized reactionproduct. The solid phase is further washed. The label in the reactionproduct is detected which indicates the presence of the antigen in thesample. The concentration of macromolecule in samples is determined asdescribed in above method.

These immunological detection methods as described above are widely usedfor detection of biologically important molecules commonly referred toas analytes. These methods are simple but have following drawbacks:

-   1. They require well-equipped laboratories and are designed for    testing samples in batches.-   2. The presence of mostly long incubation period makes the method    elongated and time consuming.-   3. The detection limit of the method is low, making the detection of    analytes present in extremely minute amount very difficult.-   4. These assays are not suitable for on-site testing or for use    under field conditions.

Dot-immunobinding assays introduced as an alternative to ELISA fordetection of antigen or antibody (Hawkes, R., et. al. AnalyticalBiochemistry 119, 142, 1982) have brought a great revolution in thefield of diagnostic. These methods commonly referred to asmembrane-based assays are similar to ELISA and only difference is thathere membrane are used as immunosorbent instead of microtitre plate ortubes. They also have competitive methods for small molecules andnon-competitive or sandwich methods for macromolecules. Here monoclonalantibody that is capable of specifically binding to the target substanceis immobilized over the membrane. In the assay, the sample to be testedis applied to the reaction membrane. If the target analyte is present inthe sample, it will bind to the immobilized receptor. Typically afterincubation step the sample was separated from the solid phase, which wasthen washed and incubated with a solution of additional polyclonalantibodies covalently, labeled with enzyme. After incubation, theunbound-labeled antibody was separated from the solid phase and theamount of labeled antibody bound to the solid phase was determined.These methods are simple, colour is visible over white background of themembrane. The method has following drawbacks:

-   1. The detection limit of the method is low and is in the range of    500 to 200 pg making the detection of antigen or antibody in test    samples difficult.-   2. The long incubation period makes the method time consuming.-   3. The low detection limit of the method has limited its application    for detection of antigen or antibody, which are present in extremely    minute amount.-   4. Membranes are delicate in nature and their handling becomes    difficult.

Detection of analyte present in low concentration requires sampleconcentration or longer incubation time to generate sufficient signalfor accurate estimation of analytes. However, because of the lowsensitivity of membrane-based assays and non-specific interference,interpretation of the results may not be accurate. Application of enzymeamplification step has been proved to be very efficient in not onlyincreasing the sensitivity but also reducing the assay time (Bates,Trends in Biotechnology. 5, 204, 1987). In these procedures, the solidphase bound primary enzyme is linked catalytically to an additionalsystem, which not only amplifies the signal but also increases thesensitivity.

Bobrow et al., (Journal of Immunological Methods, 125, 279, 1989; Ibid.137, 103, 1991) described a signal amplification system called ‘CatalyzeReporter Deposition’ (CARD) method to improve detection limit inimmunoassays using rabbit IgG as test analyte. In this method, dilutedsolution of antigen (rabbit IgG) at different concentration was appliedover nitrocellulose membrane strip as dots. It was dried, blocked with5% casein and incubated with double antibody-peroxidase conjugate. Itwas again incubated with biotin-tyramine conjugate containing 0.004%hydrogen peroxide. Membrane bound peroxidase catalyzes phenolic portionof biotin-tyramine conjugate which deposits on to the surface ofmembrane. The deposited biotin is then reacted with streptavidin labeledenzyme thereby resulting in deposition of enzymes. The net effect isthat a single HRP label is surrounded by many peroxidase molecules. Themembrane is incubated with substrate solution and due to enzymaticactivity colour develops over the membrane as dots. The intensity ofcolour is directly proportional to the amount of colour developed. Themethod also called ‘Tyramide Signal Amplification’ is flexible and canbe applied as an additional step after conventional Dot-ELISA. In thismethod, due to deposition of additional enzyme results in amplificationand thereby improving the detection limit by more than 25-fold. However,this method has following disadvantages:

-   1. The detection limit of the method with different substrate    solution depending on the enzyme label used for streptavidin is in    the range of 80 to 1 pg.-   2. The increased in sensitivity is only 30-fold.-   3. Long incubation periods required makes the method time consuming.

Recently, the applicants have developed a novel signal amplificationmethod based on catalyzed reporter deposition. The method termedSuper-CARD method utilizes synthesized electron rich proteins havingmultiple copies of phenolic group as blocking agents. After completionof conventional assay, the solid phase bound HRP oxidises the addedlabeled substrate, which deposits onto the solid phase. This depositionis markedly increased in the presence of immobilized electron richproteins, which not only amplifies the signal but also increases thesensitivity. The high specificity of the amplification reaction avoidsthe generation of any false positive signal. Direct comparison withexisting CARD methods demonstrates approximately 1.6×10⁴-foldenhancement in detection sensitivity which is much higher than that ofany other existing methods (Indian Pat. No. 1996/DEL/97; 1989/DEL/97;1991/DEL/97 and published in Journal of Immunological Methods 227,31-39, 1999; Ibid. 230, 71-86, 1999). The method offers severaladvantages:

-   1. The method is simple and easy to implement.-   2. The novel protein conjugates used as blocking agents can be    prepared from commercially available inexpensive proteins and    chemicals.-   3. The same reagents used in CARD amplification can be used.

As the membranes are very delicate and difficult to handle, numerousassay devices, in various configurations were developed for wider useunder field conditions.

The dipstick was first to be introduced, generally uses a plastic stripwith membrane containing immobilized antibody attached at one end fordipping into a solution either containing or suspected of containing theanalyte of interest. When incubated, analyte present in the sample bindsto antibody immobilized over membrane. The extent to which the analytebecomes bound to that zone can be determined with the aid of labeledreagents. Typically, the user determines the concentration of theanalyte by comparing the colour on the membrane to the colour on anexternal calibrator, such as a series of coloured plates that areprinted on a label. The colour of each plate is associated with aparticular concentration of the analyte. The colour on the plate thatmost closely approximates the colour on the dipstick provides the userwith an approximate concentration of the analyte in the test samples.The method has however several drawbacks:

-   1. Method is time consuming and often requires a number of    manipulative steps, for example, the addition and incubation of    assay reagents.-   2. It is difficult to match the colour of the plates with the colour    on the dipstick.-   3. Only one sample can be analyzed by one dipstick.-   4. The colour on the plates would not fade in proportion to the    adverse conditions affecting the colour on the dipstick. Thus, for a    particular set of reaction conditions, the comparison of results    with the colour on the plates will not give accurate result.

The Immunochromatographic test strip device constitutes an improvementover the simple dipstick. This class of devices has an absorbent stripimmobilized with receptor (antibody) near the center of a typicallyrectangular chromatography medium, e.g. filter paper, membrane andhaving an end portion for contacting a test solution. The strip having alength and width is capable of conveying fluids in a fluid flowdirection generally parallel to the length of the strip. They generallyexhibits improved sensitivity in analyte detection relative to that ofsimple dipstick devices by virtue of the analyte concentrating effectachieved by the flow of sample containing the analyte past animmobilized analyte binding zone. A sample that is suspected ofcontaining the analyte of interest is placed at or near one end of amembrane strip followed by the labeled reagent. The label reagent is ansecond antibody different from the first antibody yet it also binds withspecificity to the analyte, is prepared separately and bound to adetectable marker substance to prepare a marker-second antibody complex.The maker-second antibody complex can be premixed with sample prior toaddition to the strip or it can be added substantially simultaneouslywith the sample or it can be added after sample addition. The mixture isallowed to be carried to the opposite end of the membrane strip by aliquid phase that traverses the membrane strip by capillary action.While traversing the membrane strip, if the sample contains analyte itbinds to the receptor (either mobile or stationary phase) and the markeris also captured by the trap yielding a complex of (marker)-(secondantibody)-(analyte)-(first antibody). Because the marker is detectable,the presence of the marker can be detected by the naked eye, i.e. bymeans of colour contrasting with the chromatographic medium. Therefore,a coloured mark or the like will be left by the marker at the site atwhich the first antibody was affixed and thereby it is possible toeasily confirm the presence (or absence) of the analyte. At present,many such in vitro diagnostic kits based on immunochromatography areknown and available commercially. The methods are simple, less timeconsuming however has several drawbacks:

-   1. The method can only be used for determining the presence or    absence of substance of interest or of clinical significance. No    quantification of the analyte is possible.-   2. The materials and dimensions influence the evenness of the flow    of the detecting molecules through the assay. If the flow of liquid    is too fast, the detectable molecules are left behind and are not    accurately detected by the assay.-   3. Only single sample can be analyzed with one assay device.

Flow-through devices, which overcome some of the disadvantages of theDip-Stick and Immunochromatographic test strip devices were developedusing membrane such as nitrocellulose, glass fibre, polyester, cellulosenitrate, polyester, nylon pre-coated with an antigen. These devicesutilizes flow of fluid in a direction which is primarily transverse tothe plane of the membrane A solution containing the target analyte isdrawn through the entire membrane area by capillary action of theadsorbent material located adjacent to the membrane. Absorbent materialsuch as cellulose acetate, filter paper, porous polyethylene is capableof absorbing liquid sample in substantially greater amount than thatapplied during one test. The absorbent body provides a means to collectthe sample by providing uniform suction to deliver the sample throughthe reaction membrane down into the adsorbent body. Thus, the adsorbentbody also acts as a reservoir to hold the sample, and various reagents.Samples containing target analyte and reference standards is applied todifferent areas onto the membrane surface and absorbency of theabsorbent will draw the liquid of the sample. Thereafter, signalproducing systems capable of generating a detectable visual change onthe surface attached to a antibody having binding specificity for thetarget analyte is drawn through the membrane surface. When the colourproducing system is used, antibody conjugated to horseradish peroxidaseis exposed to the membrane surface. Subsequent exposure of the membraneto 4-Chloro-1-Naphthol substrate results in deposition of a dark bluedye on the membrane surface due to enzymatic activity. High contrastbetween the dyed and undyed portions of the membrane surface allows fordetection of analyte. Visualization of the intensity of the colour incomparison with known concentration of reference standard gives an ideaabout the amount of antigen present in the sample. The method is simpleand test can be performed under field conditions and results obtainedusually under 10 minutes.

Several analytical devices based on Flow-through principle have beendeveloped and described in patents which employs a membraneimmunoadsorbent in combination with an absorbent pad. The absorbentbody, which constitutes the fluid-receiving zone in these devices, caneither be in non-continuous contact with the membrane containingimmobilized antibody (U.S. Pat. No. 4,246,339) or in continuous contactwith membrane (U.S. Pat. Nos. 4,366,241, 4,446,232, 4,632,901 and4,727,019). Devices in which absorbent body is not in direct contactwith the membrane permit the solution containing sample and/or labeledreagents grater contact with membrane before flow of solution toabsorbent body takes place. Such non-continuous contact devices are moreefficient at utilization of sample and labeled reagents however theyrequire physical motion by assayist to bring in the flow of liquid. Thisstep can bring about error in the assay. The continuous contact devicesare less efficient in utilization of costly-labeled reagents. Thus, areagent volume substantially greater than the void volume of themembrane is required to ensure that the entire membrane has beencontacted with the solution containing reagents.

In both of these types of devices the membrane and absorbent body arecontained in a plastic housing having a top member and a bottom memberjoined together under compression to hold the membrane and absorbentbody in place and in contact with each other. In a recent U.S. Pat. No.5,885,526 to Chu (March 1999), analytical device has been slightlymodified by sealing reaction membrane and absorbent body with waterinsoluble adhesive. Liquid sample is applied to the pad by varioustechniques, and the sample drawn through the entire membrane area bycapillary action of the absorbent pad. The rate and path of fluid flowin assay device has great effect on assay results. A number of deviceshave been described in the prior art which use surfaces withspecifically arranged geometric elements to control the path and therate of fluid flow. Devices such as are described in U.S. Pat. Nos.4,426,451 and 5,922,615 utilize an arrangement in which a membrane isplaced between smooth surfaced planer sheets of a non-absorbent body inorder to contain a fluid within the membrane. Devices such as aredescribed in U.S. Pat. Nos. 4,233,029 and 4,310,399 use geometricarrangements of capillary channels to modulate the flow of fluid, suchthat fluid is directed to flow in regular geometric patterns and atcontrolled rates. Detection of analyte present in low concentrationrequires sample concentration. This has been achieved in some analyticaldevices (U.S. Pat. No. 4,818,677) by having high capacity adsorbent bodybeneath the reaction membrane, which draws larger volume of sample,added to the top of the membrane. However, because of the non-specificinterference, interpretation of the results may not be accurate. Themajor drawbacks of the developed Flow through devices till date are asfollows:

-   1. The low detection limit of analytical device has limited its    application for detection of antigen or antibody, which are present    in extremely minute amount.-   2. Analytical devices are assembled individually making the    manufacturing process complicated and costly.-   3. The use of insufficient compression to hold the membrane and    absorbent body tends sample to flow laterally during assay leading    to inaccuracy in result.-   4. Requires application of pressure to force liquid from membrane to    absorbent layer.-   5. Application of signal amplification step difficult.

OBJECTS OF THE INVENTION

The main object of the present invention is to provide an improvedhighly cost-effective, easy to manufacture analytical device based onFlow-through principle for performing rapid immunoassays for visualdetection of diagnostically important antigen or antibody. Anotherobject of the invention is to use disposable absorbent body for not onlymaking construction of the device simpler but also for application ofelements of signal amplification for attaining ultra high sensitivity.

In yet another object of the invention is to develop a simple assaymethod with the invented device which without spreading allows alwaysdownward flow of sample or costly labeled reagents efficiently, withoutapplication of any force to the absorbent body thereby overcoming abovedescribed drawbacks of the hitherto known Flow-through devices.

Novelty

The analytical device of the present invention is highly simple inconstruction compared to hitherto known devices. The materials used forthe construction are cheap and except reaction membrane, all are readilyavailable from stationary stores. The absorbent body is not fittedtogether with reaction membrane using compression or glue duringconstruction. They are provided separately with the device and arediscarded after use before proceeding for the next step. These featuresmake the present invented device completely different from hithertoknown devices. Additional absorption body are also provided if requiredby the assay protocol for addition of elements of signal amplificationsystem. The absorbent body used is a simple plane sheet of anyconventionally employed absorbent material having liquid absorptioncapability. No special geometrical arrangements in the absorbent body tocontrol the flow of fluid are required.

The assay procedure developed for performing immunoassay using theinvented device is simple and different from the known devices. Theabsorbent body is first soaked with liquid and assembled to the devicein such a way that upper surface of the absorbent body is in intimatecontact with the lower surface of the reaction membrane. The pre-wettedabsorbent body saturates the void volume of reaction membrane, whichthereby does not allow spread of sample or immunoassay reagents. Theflow of applied sample or reagent is always downwards and focusedwithout application of any force to the absorbent body therebycostly-labeled reagents can be used efficiently. The void volume of thewetted absorbent body is still sufficient to substantially fill theadditional volume of fluid introduced during assay and thereby fulfillthe function of fluid receiving zone. The wetted absorbent body controlsthe flow rate of applied liquid, which prevents spread of fluid andallows higher interaction between the target molecule and immobilizedantibody on the reaction membrane, thus increasing the sensitivity. Theimmunoassays using the invented device are simple and fast, can bequalitative or quantitative.

SUMMARY OF THE INVENTION

The present invention provides a highly cost-effective; easy tomanufacture analytical device that can be particularly used forperforming immunoassays for detection of analyte within 10 minutes withultra high sensitivity. The device of the present invention comprises asemi-rigid liquid-impervious support body and a microporous member suchas membrane containing multiple immobilized antibody spots attached atthe lower surface towards the top breadth side with water-insolubleadhesive over a semi-rigid liquid-impervious body. The device alsoincludes separate sheets of adsorbent body larger in size than membrane,which is not fixed together with membrane in the device duringmanufacture. The number of sheets of absorption body may be more thanone for further addition of elements of signal amplification systemafter discarding the first absorption body.

DETAILED DESCRIPTION OF THE INVENTION

In the method of present invention, the absorbent body is soaked influid and positioned proximal to lower surface of the reaction membraneand pressed so as to be in liquid communication with the reactionmembrane. Under these circumstances, the void volume of reactivemembrane is saturated and the distance separating the reactive membraneand the absorbent body is such that networks of capillary channels withthe absorbent material is formed. The sample or labeled reagent appliedto a well-defined area on the membrane passes always downwards withoutapplication of any external force. The applied liquid does not spreadand are focused within the limited area.

The analytical device and the method of present invention can be adoptedfor use in many different types of assay. Both conventional andultrasensitive format may be used depending upon the requirement. Assaysare rapid and batch of samples can be analyzed within 4 or 10 minutes.The device finds their greatest use not only with biological specimenbut also with industrial, environmental and food samples.

BRIEF DESCRIPTION OF THE DRAWINGS

The device of the invention can be appreciated by the figures, which areschematic drawings and are not drawn to scale.

FIG. 1 is an exploded perspective view of the components of theanalytical device.

FIG. 1A is a rectangular sheet of reaction membrane

FIG. 1B are strips of reaction membrane.

FIG. 1C is a narrow solid strip of liquid-impervious body.

FIG. 1D is a semi-rigid liquid-impervious a bottom support layer ofanalytical device.

FIG. 1E is an absorbent body, which is not attached to analytical deviceduring manufacture.

FIG. 2 (A and B) is a perspective view of the analytical deviceaccording to the present invention.

FIG. 3 (A and B) is a perspective view of the alternative analyticaldevice according to the present invention.

FIG. 4 (A and B) is a perspective view of analytical device forperforming an immunoassay in accordance with the present invention

FIG. 5 (A and B) is a perspective view of the alternative analyticaldevice for performing an immunoassay in accordance with the presentinvention

Accordingly, the present invention provides an analytical device forperforming immunoassay for the detection of analyte in a liquid samplewherein a reaction membrane which is liquid-permeable and porous andhaving an upper and lower surface, an exposed area of the said uppersurface having immobilized therein an antibody or antigen capable ofbinding to the target analyte said immobilized antibody or antigen beingconcentrated in a multiple spotted region of said upper surface.

It further comprises a semi-rigid liquid-impervious bottom support layerwherein a portion of lower surface of the said reaction membrane inbreadth corner having no immobilized antibody or antigen, is attached toupper surface of support layer by water-insoluble adhesive or tapehaving glue on both sides. Alternatively, a narrow solid strip of aliquid-impervious body placed in-between reaction membrane andsemi-rigid support layer wherein upper surface is attached to a portionof lower surface of the said reaction membrane in breadth corner andlower surface to bottom support layer by water-insoluble adhesive.

It further comprises a body of absorbent material having an uppersurface and lower surface, capable of absorbing liquid. It is not fittedtogether with reaction membrane using compression or adhesives duringmanufacture. It is provided separately with the analytical device. Theupper surface of said absorbent body extends beyond the periphery ofsaid reaction membrane but smaller than the said bottom support layer.The absorbent body is selected from the group consisting of celluloseacetate, filter paper or bathroom tissue paper. The thickness of thesaid absorbent body may range from about 0.1 to about 8 mm and more. Asingle analytical device contains more than one disposable absorbentbody, which is used for addition of elements of signal amplificationreagents.

In the analytical device of the present invention, the size andperiphery of the said reaction membrane is much smaller than the saidbottom support layer. Further, the size of the reaction membrane is notcritical and bigger size can be used in a single device to perform batchof samples. Multiple strips of the reaction membrane can be attached tosemi-rigid support layer to perform immunoassay on batch of samples. Thereaction membrane comprises nitrocellulose but other variety ofsemi-permeable membrane materials including nylon, polyvinyledinedifloride, and the like may also be used. The average diameter of themembrane is preferably in the range of about 0.22 to about 3 microns andmore preferably 0.45 microns. The loose area of the reaction membranehas immobilized thereon the antibody or antigen over entire membranesurface as multiple dots or under certain circumstances it may bedesirable to immobilize across the entire membrane surface at uniformconcentration. Further, it may be immobilize with more than one specificantibody to the membrane in the same or different areas for simultaneousdetection of multiple analyte in a sample with a single assay device.

After immobilization, unused binding sites on nitrocellulose can beblocked with suitable blocking proteins selected from the groupconsisting of casein, BSA, gelatin and like. Alternatively, forultrasensitive format vacant binding sites on nitrocellulose membraneare blocked with electron rich blocking proteins such asp-hydoxy-phenylpropionic acid-casein conjugate,p-hydroxy-phenylpropionic acid-gelatin conjugate and like forapplication of Super-CARD signal amplification.

In a further embodiment of the present invention, the bottom supportlayer with adequate mechanical strength is used and is selected from thegroup consisting of polyethylene, plastic and fiberglass. The reactionmembranes are attached over bottom support layer using water insolubleadhesive, applied only in the top 4 mm lower portion of the membrane.Alternately, adhesive tape having glue on both sides may also be used toattach membrane over bottom support layer.

In a preferred embodiment of the present invention, assembling ofabsorbent body with analytical device for performing immunoassaycomprises:

-   (a) Soaking of absorbent body with liquid followed by placing in the    analytical device in such a way that upper surface of the absorbent    body is in intimate contact with lower surface of the reaction    membrane and upper surface over bottom support layer.-   (b) The upper surface of the reaction membrane is pressed to remove    air entrapped in between lower and upper surface of reaction    membrane and absorbent body.-   (c) The void volume of reaction membrane is saturated and the    distance separating the reactive membrane and the absorbent body is    such that networks of capillary channels is formed were the two    members are in contact.-   (d) The flow of applied sample or reagent is always downwards and    focused without application of any force to the absorbent body.-   (e) The void volume of the wetted absorbent body is still sufficient    to substantially fill the additional volume of fluid introduced    during assay.

In the method of the present invention, the absorbent body is soaked indeionized distilled water, buffer and the upper surface of the reactionmembrane is pressed with small roller, rim-less small test tubes andlike. The pre-wetted absorbent body saturates the void volume ofreaction membrane, which thereby does not allow spread of sample orimmunoassay reagents. This allows the use of costly labeled reagentsefficiently. Various volumes or sample or labeled reagents ranging from10 μl to 100 μl may be used. The label reagent can be premixed withstandard or sample prior to addition to different areas of the membranein the device or it can be added after standard or sample addition. Morethan one specific antibody may also be immobilized in the same ordifferent areas for simultaneous detection of multiple analytes in asample with a single assay device.

After the addition of sample and reagents, absorbent body is discardedand the reaction membrane is washed directly over device with the helpof wash bottle. Further, for high sensitivity elements of signalamplification are further added. The signal amplification method likeSuper-CARD is applied wherein biotinylated tyramine is added directlyover reaction membrane in the device. Than a fresh pre-wetted absorbentbody is assembled and avidin-peroxidase conjugate is added and washed.This is followed by substrate solution added directly over reactionmembrane to produced colour spots within well-defined area. The exposedarea of the reaction membrane is sufficiently greater to allowvisualization of the intensity of the colour spots. Visual comparisonwith known concentration in reference standard gives semi-quantitativeestimate of the amount of antigen present in the sample Signalamplification step is not necessary for that analyte which is present inhigh concentration. The assay results can be obtained within 3 to 10minutes depending upon the format of assay used

The assay method using the device can be developed for analyteconsisting of antigens, antibodies, haptens, drugs, hormones,macromolecules, toxins, bacteria, viruses, enzymes, tumor markers,environmental pollutants, and nucleic acids.

DESCRIPTION OF THE PREFERED EMBODIMENTS

This invention provides an improved assay device, the constructionformat of, which is relatively simple, compared to prior art devices,the material cost is lower and the manufacturing process easier. Theabsorbent body is not fixed permanently with the device but is assembledduring the assay. Initially the absorbent body is wetted with fluid likewater, buffer like and positioned below reaction membrane, pressedthereby not only substantially filling the required void volume of thereactive membrane but also forming a network of capillary channels withthe absorbent body. This ensures the focused downward flow of theapplied fluid sample or reagents without any force and also does notallow outward diffusion of costly-labeled reagents. Further, theconstruction format of the device allows the discarding of usedabsorbent body with new one thereby permitting the application of signalamplification reagents for improving sensitivity of the assay.

The present invention is useful in assaying for a wide variety ofanalytes in virtually any type of sample which is liquid, which can beliquefied, or which can be suspended in liquid. The device can beincorporated in test kit and assay method using the device may bedeveloped in conventional or ultrasensitive format depending upon therequirement. The results can be obtained within 3 to 8 minutes dependingupon the format used. Analytical device will find their greatest usewith biological specimens, such as blood, serum, plasma, urine, salivaand the like. Use will also be found with industrial, environmental andfood samples.

The analyte to be detected may be virtually any compound, or othersubstance, which may be immunologically detected. That is the analyte orportion thereof will be antigenic or haptenic having at least onedeterminant site, or will be a member of a naturally-occurring bindingpair, e.g., carbohydrate and lectin, hormone and receptor, complementarynucleic acids, and the like analyte of particular interest includeantigens, antibodies, proteins, carbohydrates, haptens, drugs, hormones,macromolecules, toxins, bacteria, virus, enzymes, tumor markers, nucleicacids, and the like, although other type of substances may also bedetected. The detection of aflatoxin B1 a small molecule and human IgGare exemplified in the Examples section, hereinafter.

The analytical device of the present invention comprised of amicroporous membrane attached at one end over a semi-rigidliquid-impervious sheet. A water-insoluble adhesive is used to adhere areaction membrane having an antibody or antigen bound thereon. Thesecond member, an absorbent body, is a separate component larger in sizethan microporous membrane and is assembled during assay in such a waythat they are in intimate contact with each other at their planersurface.

The micro porous membrane of the present invention is intended toseparate and immobilize the analyte from the sample as it passes throughmembrane to the absorbent. The size of the membrane is not critical andbigger size or multiple strips can be used in a single device to performbatch of samples. However, the membrane should have sufficiently largeexposed area to allow visualization of each sample or controls on aportion thereof with sufficient excess area so that contrast between thevisual signal and the reminder of the membrane may be easily observed.The microporous membrane may be formed from a wide variety ofsemipermeable membrane materials, including nitrocellulose,polyvinylidene difluoride, nylon, paper and the like. The average porediameter of the material is usually not critical, although materialshaving particular pore diameter may be selected for particular assay.However, the porosity of the membrane has a large influence on the flowrate of the liquid and sensitivity of the assay. The larger the poressize of the membrane, the faster the flow rate for a given liquid. Asthe flow rate increases, the interaction time available between thetarget molecule in the sample and the antibody immobilized on thereaction membrane decreases, thus decreasing assay sensitivity. For mostassays, the porosity of membrane is preferably in the range of about0.22 to about 3 microns. For most applications, specific bindingsubstances like antibody will be bound to the membrane to facilitateseparation of the analyte of interest.

An important preferred embodiment of the invention is the selection ofnitrocellulose membrane as the carrier material. This has considerableadvantage because it has a natural ability to bind proteins withoutrequiring prior sensitization. Such specific binding substance areusually antibodies capable of binding antigens and haptens, althoughantigens, hormone receptors, lectin, polysaccharides, nucleic acids, andother natural receptors and legends may also find use. Methods forbinding the specific binding substances to the nitrocellulose membraneare well known and amply described in the scientific and patentliterature. Unused binding sites on the nitrocellulose can therefore beblocked with blocking proteins such as casein, BSA or gelatin. Electronrich blocking proteins such as p-hydroxyphenylpropionic acid-casein,p-hydroxyphenylpropionic acid-gelatin conjugate are used for blockingvacant sites of the nitrocellulose membrane for ultrasensitive detectionof analyte for application of Super-Card signal amplification (J.Immunological Methods 227, 31, 1999).

In a preferred embodiment of the present invention the specific bindingsubstances on uniform concentration is immobilized as multiple spotsover entire membrane surface or under certain circumstances it may bedesirable to bind the substances across the entire membrane surface. Thespot approach is preferred, as sample and labeled reagents isconcentrated at a single region thereby providing a more distinctive endproduct signal. In a further particularly preferred embodiment, it maybe desirable to sometimes bind more than one specific binding substanceto the membrane in the same or different areas, for simultaneousdetection of multiple analytes in a sample with a single assay device.

The analytical device of the present invention further comprises asemi-rigid liquid-impervious bottom support layer. The reaction membraneis placed over semi-rigid support layer and one end is attached alongbreadth side through lower surface. The size of the bottom support layeris much larger than reactive membrane and absorbent body. The bottomsupport layer is typically comprised of plastic, polyethylene, polyesterand like. The selection of proper materials with adequate mechanicalstrength as the supporting backing for the device is important.Undesirable bending of the assay device may occur if a week backing orinadequate mechanical strength of bottom support layer is used. As hasalready been noted, typical prior art analytical devices are comprisedof top and bottom housing members which are fitted together so as tohold the reaction membrane and absorbent body in place usingcompression. Because the reaction membrane and absorbent bodies are twoindependent components of the device and absorbent body is placed onlyduring the assay, there is no need to use fitted or otherwise sealed topand bottom housing members. Bottom support layer supports the reactionmembrane during transit and handling of membrane becomes easier duringassay. Materials used for bottom support layer could also be used toprovide top support layer to cover the membrane, although it may bedifferent.

In yet another aspect of the present invention, multiple microporousmembrane strips or in one piece having multiple reactive sites can beattached over solid support layer for analyzing batch of samples.Alternatively, in between the membranes and solid support layer a narrowsolid strip typically comprised of any water-impervious material may beattached to provide sufficient space for the absorbent body to be placedbeneath the reaction membrane during the assay. The same material assolid support layer having the thickness similar or higher thanabsorbent material may be used. After the device has been constructed,the top solid support layer may be labeled with a labeling or bar codedevice to indicate the type of test for which the device is to be used.

In a further embodiment of the inventive device, absorption body is notfitted together with reaction membrane using compression or glue duringmanufacture. They are provided separately and are discarded after usebefore proceeding for the next step. Additional absorption body are alsoprovided if required by the assay protocol. In a further embodiment ofthe analytical device, additional absorption body is used according toabove described method for addition of elements of signal amplificationsystem.

The primary requirement of the absorbent solid material is that it iscapable of absorbing liquid. Any conventionally employed absorbentmaterial that has liquid absorption capability can be used in thepresent invention. Useful known materials includes cellulose acetatefilters, polyester or other such materials. Layers of commerciallyavailable filter paper or bathroom tissue paper can be used. Thethickness of the absorbent body (i.e. “side-wall”, which is the distancebetween the upper and lower surfaces of the absorbent material) can varydepending upon the void volume needed for a given immunoassay.Typically, the thickness ranging from about 0.1 mm to about 8 mm, andmore. The surface area of the absorbent body is usually greater thanthat of the reaction membrane but smaller than the bottom support layer.In a preferred embodiment of the present invention absorbent body usedis approximately five times the size of the reaction membrane. In theexemplary embodiment, the absorbent body comprises Whatman filter paperNo 3.

In the method of the present invention, absorbent body provided with theanalytical device is first soaked with excess liquid such as deionizedwater, buffer and like. The wetted absorbent body is assembled with theanalytical device in such a way that upper surface of the absorbent bodyis in intimate contact with lower surface of the reaction membrane andlower surface is over bottom support layer. The upper surface of thereaction membrane is than pressed with a small roller or rimless testtube in such a way that air entrapped in between lower and upper surfaceof reaction membrane and absorbent body is removed. Under thesecircumstances, the void volume of reactive membrane is saturated and thedistance separating the reactive membrane and the absorbent body is suchthat networks of capillary channels is formed were the two members arein contact. The wetted reaction membrane and absorbent body do not allowthe fluid of sample and reagents to flow sideways across the reactionmembrane into the absorbent body. In the method of the presentinvention, the flow of the sample or reagent is always downwards andfocused without application of any force to the absorbent body therebycostly-labeled reagents can be used efficiently. The void volume of thewetted absorbent body is still sufficient to substantially fill theadditional volume of fluid introduced during assay and thereby fulfillthe function of fluid receiving zone. The wetting of the absorbent bodyhas large influence on the flow rate of the liquid and sensitivity ofthe assay. Excess wetting of the absorbent body reduces the flow ratethereby allowing the fluid to spread. If the wetting is less, the flowrate increases, which decreases the interaction between the targetmolecule in the sample and the immobilized antibody on the reactionmembrane, thus decreasing the sensitivity.

The immunoassays that use the analytical device of the present inventioncan be very simple and fast, can be qualitative or quantitative. Manydifferent types of immunoassays, known in the art can be performed usingthese analytical devices. The immunoassay format depends on the type ofanalyte to be detected.

Various detection reagents can be used to further simplify and improveassay sensitivity. Direct labels such as gold sols and dye solutions canalso be used. Again methods are already known in the art. Assay methodusing the device may be in conventional or ultrasensitive formatdepending upon the requirement. The single absorbent body is used foraddition of standards or sample and label reagent. The label reagent canbe premixed with standard or sample prior to addition to different areasof the membrane in the device or it can be added after standard orsample addition. In ultrasensitive format the used absorbent body isremoved after the convention immunoassay and membrane surface in theanalytical device is directly washed. This is followed by addition ofsignal amplification reagent directly and or using a new absorbent bodyprovided with the device. For example, for Super-CARD signalamplification method, a solution of biotinylated tyramine solution isadded directly over membrane without absorbent body and washed withwashing buffer. Then a new absorbent body is assembled according to themethod described above and Avidin-peroxidase is added over reactionmembrane. Visual comparison of the intensity of the colour of the spotswith those of known concentration in reference standard givessemi-quantitative estimate of the amount of antigen present in thesample.

All cited references are incorporated herein by references in theirentireties. The following examples and drawings are for illustrativepurpose only and are not to be construed as limiting the scope of theinvention in any manner.

EXAMPLE 1

Conventional Assay for Aflatoxin B₁ using Present Invention

-   A. Antibody to Aflatoxin B₁. The immunogen, aflatoxin    B₁—O-Carboxymethyloxime-BSA conjugate was prepared according to a    standard method using stoichiometry of 20:1 aflatoxin B₁ derivative:    protein. The immunogen was injected into rabbits together with    Freund's adjuvant and a highly specific antiserum was obtained. The    gamma globulin fraction of this antiserum was purified by repeated    precipitation with ammonium sulfate followed by dialysis against    phosphate buffer saline. It was than passed through a BSA-Sepharose    4B immunosorbent column to remove anti-BSA antibody.-   B. Test Device Preparation. Schleicher & Schuell nitrocellulose    membrane having pore size of 0.45 micron is cut to a rectangular    piece (14 mm. times. 70 mm) and marked with pencil 14×14 mm (FIG.    1A). The membrane can also be cut in strips (14×70 mm) and marked    with pencil 14×14 mm (FIG. 1B). The membrane is then soaked in    blotting buffer (Tris-HCl, 20 mM, pH 8.0 containing 0.9% NaCl) for 5    min with gentle shaking. The strips were than semi-dried and spotted    with dispenser (Hamilton) at multiple sites at a distance of 1.4 cm    with 5 μl of a rabbit antibody to aflatoxin B₁ diluted 100-fold in    Tris-buffered saline (0.05M, pH 8.0). The top 14 mm square in the    breadth side of the rectangular membrane was not spotted for    attachment to semi-rigid material. After drying the membrane at room    temperature for 15 min it was further dried by incubating at 37° C.    for 30 min. The vacant sites of the membrane strip were blocked by    incubating with 0.4% casein in carbonate-bicarbonate buffer (0.05M,    pH 9.6) for 1 hr. at 22 to 26° C. with gentle shaking. The membrane    strips were than washed three times with washing buffer (Tris-HCl    buffer, 20 mM, pH 8.0, containing 29 g/l NaCl and 0.5% Tween 20).    Finally membranes were rinsed with 0.1% thimerosal and than dried at    room temperature for 15 min followed by at 37° C. for 30 min. These    membranes can be stored in a desecrator till use.-    To assemble the test device, a semi-rigid material such as    polyethylene sheet (140×120 mm) with thickness about 0.005 to 0.030    inches was taken (FIG. 1D). In the unspotted corner top of the    membrane, a 4 mm layer of water-insoluble adhesive is applied. The    membrane is than placed at a distance of 15 mm from the top in the    breadth wise over rectangular polyethylene sheet (FIG. 2). Adhesive    tape having glue on the both sides may also be used to attach    membrane.-    Alternatively, a narrow solid strip of a liquid-impervious body    placed in-between membrane and semi-rigid support layer may be used    wherein upper surface is attached to a portion of lower surface of    the said membrane in breadth corner and lower surface to bottom    support layer by water-insoluble adhesive (FIG. 3).-    One rectangular piece of filter paper (Whatman No. 3) measuring    125×110 mm. (FIG. 1E) is cut from the filter paper sheet for use    during the assay. This analytical device can be stored in a    desecrator.-   C. Enzyme Conjugate Preparation. Aflatoxin B₁—O-carboxymethyl    oxime-Horseradish peroxidase conjugate was prepared according to a    standard method using stoichiometry of 5:1 aflatoxin B₁ derivative:    peroxidase. It was purified by dialysis followed by chromatography    on Sephadex G-50. After the addition of BSA, 20 g/l, working    solution of enzyme conjugate (1:100) was prepared in phosphate    buffer (0.05M, pH 7.6 containing per litre 0.9% NaCl, 0.4% BSA and    0.01% thimerosal).-   D. Sample Preparation. The infected seeds were soaked in water for 1    hr at 22 to 26° C. and dried on filter paper. The seeds (1 gm) was    homogenized in methanol (10 ml) and centrifuged at 10,000 rpm for 15    min. The supernatant was used in the assay by diluting 1000-fold    with assay buffer (Tris-HCl buffer, 0.05M, pH 8.0 containing 0.9%    NaCl, 0.02% BSA and 0.01% thimerosal).-   E. Aflatoxin B₁ Standards. Aflatoxin B₁ stock standard solution    (0.25 mg/ml in acetonitrile) was stored in −20° C. and working    standard solution (0, 25 pg, 50 pg, and 100 pg/25 μl) was prepared    by diluting with assay buffer.-   F. Substrate solution. 3,3′-Diaminobenzidine (15 mg) and Immidazole    (201 mg) was dissolved in 30 ml of Tris-HCl buffer (0.05M, pH 8.0)    and 0.001% H₂ O₂ was added. The solution was stored in the dark till    use.-   G. Procedure for Testing the Presence of Aflatoxin B₁ in Samples    (Two-step). Rectangular piece of filter paper (FIG. 1E) was wetted    with distilled water and placed in between membrane and polyethylene    sheet of the Test device (FIG. 4 or 5). Air entrapped in-between    membrane and filter paper was removed by rotating a rimless glass    tube over membrane strip. Standard or sample (25 μl) was added    slowly over different antibody spotted area of the membrane. The    solution is immediately absorbed due to capillary action created by    filter paper. Now, over each spotted area where standard or samples    have been added, 25 μl of AFB₁-HRP conjugate (1:1000) in assay    buffer was added. Filter paper is removed and membrane is thoroughly    washed with wash buffer. Substrate solution (DAB) is added over    membrane and incubated for 1 min and washed with water.    Visualization of the intensity of the colour spots in comparison    with known concentration in reference standard gives an idea about    the amount of antigen present in the sample. The assay sensitivity    is measured to be 20 pg/25 μl aflatoxin B₁.

EXAMPLE 2

The same procedures as Example 1(A), 1(B), 1(C), 1(D), 1(E), 1(F) isfollowed except that in 1(F) standard 50, 100 and 200 pg/25 μl wereused. Standard or samples is mixed in equal proportion with AFB₁-HRPconjugate (1:500) and 25 μl was added over AFB₁ antibody spotted area.The results obtained in this One-step method were almost similar to thatof Two-step.

EXAMPLE 3

The same procedure as Example 2 is followed except that standard orsamples is mixed in equal proportions with 1000-fold AFB₁-HRP conjugateand 25 μl of each mixture were added twice over AFB₁ antibody spottedarea. The results obtained in this method were almost similar to thoseobtained in Examples 1 and 2.

EXAMPLE 4

Ultrasensitive Assay for Aflatoxin B₁ using the Present Invention

-   A. Preparation of 3-(p-hydroxyphenyl) propionic acid-casein    conjugate (p-OH-PPA-casein). Casein (1 gram) dissolved in 30 ml of    sodium bicarbonate (150 mmol/l) was added dropwise under stirring    300 mg of p-OH-PPA-N-hydoxysuccinimide dissolved in dry    dimethylformamide. The reaction mixtures were stirred continuously    at room temperature for 3 hours and than dialyzed extensively    against distilled water and subsequently against phosphate buffer    (20 mmol/l, pH 7.6). The p-OH-PPA-casein conjugate obtained were    centrifuged to remove the slight turbidity and then lyophilized.-   B. Preparation of biotinylated tyramine. A solution of biotin (100    mg) in a mixture of dry dimethylformamide and dimethylsulfoxide    (1:2.3) was treated with N-hydroxysuccinimide (70 mg) and    dicyclohexylcarbodiimide (120 mg) overnight at 4° C. The activated    ester solution was filtered to remove urea and added to a solution    of tyramine hydrochloride (60 mg) in 1 ml of dimethylformamide. The    reaction mixture was stirred overnight in the presence of powered    solid sodium carbonate and filtered. The resulting solution (3 ml)    was aliquoted and stored at −20° C. A working solution (200 μmol/l    in distilled water) was prepared and stored at 4° C. In assay    biotinylated tyramine working solution was diluted 2-fold with    borate buffer (0.2 M, pH 8.0 containing 0.008% H₂O₂) and used    directly.-   C. Test Device Preparation. Schleicher & Schuell nitrocellulose    membrane having pore size of 0.45 micron is cut to a rectangular    piece (14 mm. times. 70 mm) and marked with pencil 14×14 mm (FIG.    1A). The membrane can also be cut in strips (14×70 mm) and marked    with pencil at a distance of 14 mm (FIG. 1B). The membrane is soaked    in blotting buffer (Tris-HCl, 20 mM, pH 8.0 containing 0.9% NaCl)    for 5 min with gentle shaking. The strips were than semi-dried and    spotted with dispenser (Hamilton) at multiple sites at a middle of    14 mm square with 5 μl of a rabbit antibody to aflatoxin B₁ diluted    2500-fold in Tris-buffered saline (0.05M, pH 8.0) supplemented with    25 μg/ml BSA. The top 14 mm square in the breadth side of the    rectangular membrane was not spotted for attachment to semi-rigid    material. After drying the membrane at room temperature for 15 min    it was further dried by incubating at 37° C. for 30 min. The vacant    sites of the membrane strip were blocked by incubating with 0.2%    p-OH-PPA-casein in carbonate-bicarbonate buffer (0.05M, pH 9.6) for    1 hr. at 22 to 26° C. with gentle shaking. The membrane strips were    than washed three times with washing buffer (Tris-HCl buffer, 20 mM,    pH 8.0, containing 29 g/l NaCl and 0.5% Tween 20). Finally membranes    were rinsed with 0.1% thimerosal and than dried at room temperature    for 15 min followed by at 37° C. for 30 min. These membranes can be    stored in a desecrator till use.-    To assemble the test device, a semi-rigid material such as    polyethylene sheet (140×120 mm) with thickness about 0.005 to 0.030    inches was taken (FIG. 1D). In the unspotted corner top of the    membrane, a 4 mm layer of water-insoluble adhesive is applied. The    membrane is than placed at a distance of 15 mm from the top over    rectangular polyethylene sheet (FIG. 2). Adhesive tape having glue    on the both sides may also be used to attach membrane.-    Alternatively, a narrow solid strip of a liquid-impervious body    placed in-between membrane and semi-rigid support layer may be used    wherein upper surface is attached to a portion of lower surface of    the said membrane in breadth corner and lower surface to bottom    support layer by water-insoluble adhesive (FIG. 3).-    One rectangular piece of filter paper (Whatman No. 3) measuring    125×110 mm. (FIG. 1E) is cut from the filter paper sheet for use    during the assay. This analytical device can be stored in a    desecrator.-   D. Sample Preparation. The infected seeds were soaked in water for 1    hr at 22 to 26° C. and dried on filter paper. The seeds (1 gm) was    homogenized in methanol (10 ml) and centrifuged at 10,000 rpm for 15    min. The supernatant was used in the assay by diluting 20,000-fold    with assay buffer (Tris-HCl buffer, 0.05M, pH 8.0 containing 0.9%    NaCl, 0.02% BSA and 0.01% thimerosal).-   E. Aflatoxin B₁ Standards. Aflatoxin B₁ stock standard solution    (0.25 mg/ml in acetonitrile) was stored in −20° C. and working    standard solution (0, 1 pg, 5 pg, and 10 pg/25 μl) was prepared by    diluting with assay buffer.-   F. Substrate solution. 4-chloro-1-naphthol (15 mg) was dissolved in    6 ml methanol and diluted to 30 ml with Tris-HCl buffer (0.1 M, pH    8.0). The solution was stored in the dark after addition of 0.001%    H₂O₂.-   G. Procedure for Testing the Presence of Aflatoxin B₁ in Samples    (Two-step). Rectangular piece of filter paper was wetted with    distilled water and placed in between membrane and polyethylene    sheet of the Test device (FIG. 4 or 5). Air entrapped in-between    membrane and filter paper was removed by rotating a rimless glass    tube over membrane strip. Standard or sample (25 μl) was added    slowly over different antibody spotted area of the membrane. The    solution is immediately absorbed due to capillary action created by    filter paper. Now, over each spotted area where standard or samples    have been added, 25 μl of AFB₁-HRP conjugate (1:20,000) in assay    buffer was added. Filter paper is removed and membrane is thoroughly    washed with washing buffer. For amplification, membranes were    treated with biotinylated tyramine solution at room temperature for    2 min. Membranes were again washed with washing buffer. Then again    rectangular piece of filter paper was wetted with distilled water    and placed in between membrane and polyethylene sheet of the Test    device. Air entrapped in-between membrane and filter paper was    removed by rotating a rimless glass tube over membrane strip.    Avidin-HRP conjugate (purchased from Sigma, St. Louis, USA) was    diluted 500-fold with assay buffer and 25 μl was added over each    anti-AFB₁ spotted area. Filter paper is removed and membrane is    thoroughly washed with wash buffer. Substrate solution (4CN) is    added over membrane and incubated for 2 min and washed with water.    Visualization of the intensity of the colour spots in comparison    with known concentration in reference standard gives an idea about    the amount of antigen present in the sample. The assay sensitivity    is measured to be 0.5 pg/25 μl aflatoxin B₁.

EXAMPLE 5

The same procedures as Example 4(A), 4(B), 4(C), 4(D), 4(E), 4(F). 4(G)is followed except that in 4(G) standard 2, 10 and 20 pg/25 μl was used.Standard or samples is mixed in equal proportion with AFB₁-HRP conjugate(1:10,000) and 25 μl was added over AFB₁ antibody spotted area. Theresults obtained in this One-step method showed slightly highersensitivity than that of Two-step method.

EXAMPLE 6

Ultrasensitive Assay for Human IgG using the Present Invention

Analytical device was assembled as described in Example 4 except thatmembrane was spotted with 5 μl of 1:100 Protein A solution (Stock 0.4mg/ml) in sodium phosphate buffer (10 mM, pH 7.6 containing 137 mM NaCl,0.02% KCl) with Dilutor (Hemilton). The reagent p-OH-PPA-casein,biotinylated tyramine and substrate were same as described in Example 4.

-   A. Human IgG standards. Human IgG stock standard solution (0.25    mg/ml in assay buffer) was diluted with assay buffer to prepare    working standards: 62.5, 125, 250, 500 and 1000 ng/25 μl.-   B. Assay procedure. The same assay procedure as described in Example    4G is followed. Human IgG standards (25 μl) was added to the    reaction membrane and allowed to completely absorb. Then, 25 μl of    Protein A-HRP conjugate (Sigma, USA) diluted 1:8000 in Tris-HCl    buffer (50 mM, pH 8.0 containing 0.4% BSA) was added and allowed to    completely absorb. Washing buffer (1 ml) was added to the reaction    membrane and then washed with washing buffer to remove unbound    reagents from the membrane. Now the signal amplification reagents    are added as described in Example 4G. A sample containing unknown    quantities of IgG was determined (semi-quantitative) by comparing to    the standard curve. This demonstrates that the analytical device can    be used to detect low concentration of IgG.

EXAMPLE 7

Analytical device was assembled as described in Example 6. Serialdilution of normal human serum (1:500 to 1:32,000) was made in sodiumphosphate buffer saline containing 0.4% BSA. A 25 μl of each dilution ofhuman serum was added to the reaction membrane and allowed to completelyabsorb. A 25 μl of the Protein A-HRP conjugate solution (1:8000)described in Example 6 was added to the reaction membrane and allowed tocompletely absorb. The remainder of the assay is similar to thatdescribed in Example 6. At the end of the assay, a purple spot wasvisible on the analytical device at a 1:16,000 dilution of normal humanserum, indicating that IgG was present and detectable at that dilutionusing the analytical device and the above assay procedures.

Advantages

-   i. Analytical device is highly cost-effective and easy to    manufacture.-   ii. The materials used for the construction are cheap and except    reaction membrane, all are readily available from stationary stores.-   iii. Absorbent body is not fitted together with reaction membrane    using compression or glue during manufacture and are provided    separately.-   iv. Absorbent body is assembled during the assay and is discarded    after use before proceeding for the next step.-   v. Additional absorbent body are also provided if required by the    assay protocol for addition of elements of signal amplification    system.-   vi. The method developed for performing immunoassay using the device    is simple and allows the flow of the sample and costly-labeled    reagents always downwards and focussed without application of any    force to the absorbent body.-   vii. The method is useful in assaying wide variety of analytes.-   viii. Assays are non-instrumental and can be used under field    conditions.-   ix. Visualization of the intensity of the colour spots in comparison    with known concentration in reference standard gives an idea about    the amount of antigen present in the sample.-   x. Sensitivity of the method can be increased by signal    amplification.-   xi. Results can be obtained within 3 to 8 minutes depending on the    immunoassay format used.-   xii. Batch of samples can be analyzed.-   xiii. Direct labels such as gold sols and dye solutions can be used.-   xiv. Skilled personal not required to perform the assay.-   xv. Quantitative results are possible by using Gel-Documentation    system.

1-21. (canceled)
 22. An method for assembling an absorbent body of ananalytical device comprising a liquid-permeable porous reaction membranehaving an upper and a lower surface, an exposed area of the uppersurface having immobilized thereupon an antibody or antigen capable ofbinding to the target analyte, wherein said immobilized antibody orantigen being concentrated in multiple spotted regions of the said uppersurface, and a portion of the lower surface of the reaction membrane inbreadth corner has no immobilized antigen or antibody; a semi-rigidliquid impervious bottom support layer attached to the lower surface ofthe reaction membrane at the breadth corner by water insoluble adhesiveor tape having glue on both sides; and a body of absorbent materialhaving an upper surface and a lower surface, capable of absorbingliquid, wherein the body of absorbent material is provided separatelyfrom the analytical device and in use, the absorbent material ispre-wetted with a liquid and is placed between and in contact with thereaction membrane, which is above the body of the absorbent material andthe bottom support layer, which is below the body of the absorbentmaterial and the body of absorbent material is capable of absorbingliquid and is larger is size than the reaction membrane, wherein theabsorbent body is assembled by: (a) soaking the absorbent body withliquid and assembled in such a way that upper surface of the absorbentbody is in intimate contact with lower surface of the reaction membraneand upper surface over bottom support layer, (b) removing the entrappedair in between lower and upper surface of reaction membrane andabsorbent body by pressing the upper surface of the reaction membrane,(c) the void volume of reaction membrane is saturated and the distanceseparating the reactive membrane and the absorbent body is such thatnetworks of capillary channels is formed were the two members are incontact, (d) the flow of applied sample or reagent is always downwardsand focused without application of any force to the absorbent body, and(e) the void volume of the wetted absorbent body is still sufficient tosubstantially fill the additional volume of fluid introduced duringassay.
 23. The method as claimed in claim 22 wherein, absorbent body issoaked in deionized distilled water, buffer and other similar materials.24. The method as claimed in claim 22 wherein, the upper surface of thereaction membrane is pressed with small roller, rim-less small test-tubeand other similar tools.
 25. The method as claimed in claim 22 wherein,pre-wetted absorbent body saturates the void volume of reactionmembrane, which thereby does not allow spread of sample or immunoassayreagents.
 26. The method as claimed in claim 22 wherein, thecostly-labeled reagents are used efficiently.
 27. The method as claimedin claim 22 wherein, 10 to 100 μl of sample or labeled reagents areused.
 28. The method as claimed in claim 22 wherein, multiple of 10 to100 μl of sample or labeled reagents are applied.
 29. The method asclaimed in claim 22 wherein, the label reagent is premixed with standardor sample prior to addition to different areas of the membrane in thedevice or it can be added after standard or sample addition.
 30. Themethod as claimed in claim 22 wherein, more than one specific antibodyis immobilized in the same or different areas for simultaneous detectionof multiple analytes in a sample with a single assay device.
 31. Themethod as claimed in claim 22 wherein, after application of samples andreagents absorbent body is discarded.
 32. The method as claimed in claim22 wherein, the reaction membrane is washed directly over device withthe help of wash bottle.
 33. The method as claimed in claim 22 wherein,for high sensitivity elements of signal amplification are further added.34. The method as claimed in claim 22 wherein, the signal amplificationmethod like Super-CARD is applied.
 35. The method as claimed in claim 22wherein, biotinylated tyramine is added directly over reaction membranein the device.
 36. The method as claimed in claim 22 wherein, a freshpre-wetted absorbent body is assembled and avidin-peroxidase conjugateis added.
 37. The method as claimed in claim 36 wherein, the substratesolution added directly over reaction membrane produced color spotswithin well-defined area.
 38. The method as claimed in claim 37 wherein,the exposed area of the reaction membrane is sufficiently greater toallow visualization of the intensity of the color spots.
 39. The methodas claimed in claim 38 wherein, visual comparison with knownconcentration in reference standard gives semi-quantitative estimate ofthe amount of antigen present in the sample.
 40. The method as claimedin claim 36 wherein, the substrate solution is added directly withoutapplication of signal amplification.
 41. The method as claimed in claim36 wherein, signal amplification step is not necessary for that analytewhich is present in high concentration.
 42. The method as claimed inclaim 22 wherein, the assay results is obtained within 3 to 10 minutes.43. The method as claimed in claim 22 wherein, the analyte is selectedfrom the group consisting of antigens, antibodies, haptens, drugs,hormones, macromolecules, toxins, bacteria, viruses, enzymes, tumormarkers, environmental pollutants, nucleic acids and other naturalreceptors.